A clonogenic assay is a cell biology technique for studying the effectiveness of specific agents. The experimental work described in this thesis was carried out under the supervision of professor martin clynes. Possible explanations could be that as mart1 and gp100 are melanocyte differentiation ag, clonogenic agnonexpressing cells would be spared by immune effectors, or that clonogenic cells would be intrinsically resistant to. In vitro and in vivo assays of each epc colonyforming unit cell revealed a differentiation hierarchy from small epc to large epc colonies, indicating a primitive epc stage with highly proliferative activity and a definitive epc stage with vasculogenic properties, respectively.
In vivo drug sensitivity assay of clonogenic human. Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The number of lung colonies is a measure of the number of clonogenic tumor cells in the injected suspension. Here we developed an in vivo in vitro clonogenic assay to characterize the survival of neural stem cells after exposure to ionizing radiation. In the traditional soft agar colony formation assay, cells are grown in a layer of soft.
The clonogenic assay is the method of choice to determine cell reproductive death after in vitro irradiation treatment. National cell and tissue culture centre bioresearch ireland, school of biological sciences. The equivalence to a clonogenic survival assay with its. A clonogenic assay is a cell biology technique for studying the effectiveness of specific agents on the survival and proliferation of cells. Determination of cell survival after irradiation via clonogenic assay. B hif1 activation with molidustat impairs the clonogenic potential of mdamb231 cells. Two cell lines are used for cytotoxicity determination. In vitro cytotoxicity mtt assay in vero, hepg2 and mcf 7. Therefore, we investigated if proton irradiation in a. Radiosensitivity of head and neck cancer cells in vitro.
In vitro and in vivo assays of each epc colonyforming unit cell revealed a. Our ex vivo models include freshly isolated cells from pdx tumors utilized in assays immediately on the day of isolation. A guidance document on good in vitro method practices givimp. A wide range of in vitro assays techniques have been developed to evaluate each hallmark feature of cancer and selection of a particular in vitro assay or technique mainly depends on the. Mart1 and gp100 are prototypical melanoma antigen ag, but their clinical use as vaccines or as targets of cytotoxic lymphocytes achieved modest success. The proportion of viable cells in a cell population can be estimated in. Chemosensitivity assays include, but are not limited to, the following. The most widely used system is the clonogenic assay, which has demonstrated some clinical predictivity. Experimental survey of nonclonogenic viability assays for. A test for comparision of cell survival curves and an anova test for experimental twoway designs are provided. Using nk92 effector cells as example, results from rtca potency assay are very well correlated with end point data from imagebased assays as. Pdf electrical woundhealing assay for cells in vitro. Cells were grown in the presence or absence of molidustat 1050. Clonogenic assay of cells in vitro nature protocols.
It is frequently used in cancer research laboratories to determine the effect of drugs or radiation on proliferating tumor cells as well as for titration of cellkilling particles ckps in virus stocks. In vitro ex vivo assays for cancer pharmacology factsheet. Injection into lewis x bn f, hybrid lbn rats resulted in a log. In cancer treatment, cell viability is a basic and important parameter for predicting radio sensitivity. Methodology open access determination of cell survival. Sunitinib reduces acute myeloid leukemia clonogenic cells. As the cells are removed from the living in vivo environment and subjected to experimental manipulations in the culture systems in vitro. Clonogenic cell survival assay clonogenic survival assay was performed on the basis of a standard procedure with slight modification 8. Heterogeneity of human metastatic clones by in vitro. There have been many attempts to design in vitro systems to determine drug response of tumors. Methodological development of a clonogenic assay to. The assay is less common to study survival of cancer cells after irradiation, in particular when the mtt assay is performed for studying proliferation of treated cells.
Applications of highthroughput clonogenic survival assays. Cells were isolated from the rat cervical spinal cord and plated as single cell suspensions in defined medium containing epidermal growth factor and basic fibroblast growth factor. Realtime cellimpedance sensing assay as an alternative. The motivating factor in developing this method was the need for an in vitro assay for clonogenic epidermal and hair follicle stem cells 1, 2. The assay essentially tests every cell in the population. Complex readouts which capture multipleall genes, proteins. In vitro clonogenic assays have been developed and widely used since many years to investigate the proliferation and the differentiation both of pluripotent haemopoietic stem cells phsc and of the different progenitors of blood cell lineages. A cell survival curve is therefore defined as a relationship between the dose of the agent used to produce an insult and the fraction of cells retaining their ability to reproduce. Although multiple studies of clonogenic assays on cancer cell lines have been published, the robustness of this technique has not been examined by comparative analysis of data from different studies. Hif1 stabilization exerts anticancer effects in breast.
The clonogenic cell survival assay determines the ability of a cell to proliferate indefinitely, thereby retaining its reproductive ability to form a large colony or a clone. Thereafter, cell viability was assessed using mtt assay. Pdf clonogenic assay of cells in vitro nicolaas klaas. Mart1 and gp100expressing and nonexpressing melanoma. Evaluation of mtt and trypan blue assays for radiationinduced cell. Dishes were incubated to allow colony formation for 1014 days. A clonogenic survival assay of neural stem cells in rat. The assay is less common to study survival of cancer cells after irradiation, in particular when the mtt assay is. The assay essentially tests every cell in the population for its ability to undergo unlimited division. In vitro assays and techniques utilized in anticancer drug. Clonogenic assays are commonly used to investigate survival of irradiated cancer cells, whereas mtt assays are well known to study chemosensitivity or toxicity of drugs in human tumor cell lines. In addition, the combination of the radiosensitizing agent gemcitabine and radiation was investigated with both. The assay has been extensively used in studies both of individual patients response to chemotherapy and for screening new agents.
The outcome is the correlation between deposited radiation dose and biological endpoint. In vitro biological response of cancer and normal tissue. Franken na1, rodermond hm, stap j, haveman j, van bree c. Clonogenic assay or colony formation assay is extensively used to measure in vitro cell survival based on the capacity of a single cell to grow into. My project is the validation of the clonogenic assay, which is used to study the effects of irradiation in glioblastoma 1. Clonogenic assays are the gold standard for determining radiosensitivity, which governs tumor response to radiation therapy. The human tumor stem cell clonogenic assay htca is a soft agar system designed for growing fresh human tumor specimens in vitro. The assay essentially tests every cell in the population for its ability to undergo.
In vitro immunotherapy potency assays using realtime cell. Biomarkers and functional tests to confirm the required cell function state 55. The colony is defined to consist of at least 50 cells. Isolation of mouse epidermal keratinocytes and their in. Cells were incubated with increasing doses of molidustat for 48 h. A 96well plate clonogenic cell assay for squamous cell carcinoma. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability capacity of cells to produce progeny. Clonogenic assay or colony formation assay cfa is an in vitro cell. Disc assay differential staining cytotoxicity assay an in vitro study for hematologic malignancies. A comparative pharmacometabolomic study of glutaminase. Read clonogenic assay and in vitro chemosensitivity testing of human urologic malignancies, cancer on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Mtt readings are proportional to the number of cells invitro, at least in the phase of. The clonogenic assay is an in vitro cell survival assay that evaluates all modalities of cell death based on the ability of a single cell to grow into a colony.
The colorimetric sulforhodamine b srb assay was compared to the clonogenic assay for radiosensitivity testing in two lung cancer cell lines a549, h292, one colon cancer cell line ht29 and one breast cancer cell line mcf7. The cells are from a line of u87, which are cancerous human glial cells in vitro. A subline of the brown norway myeloid leukemia in the. Ideally, an in vitro tumor sensitivity assay must be reliable, sensitive, and resemble the 3d, in vivo environment such as culturing in collagen gel or soft agar. The difference in these assays is determined by the processing method.
Results of rapid cell viability assays were experimentally compared in order to reveal the most suitable test for in vitro investigations of the combination of photodynamic therapy pdt with chemotherapeutic drugs. Malignant mononuclear cells are incubated with specific. Determination of cell survival after irradiation via. Cell radiosensitivity can be examined by performing a clonogenic survival assay in vitro. Tumors are complex systems consisting of heterogeneous cancer cells as well as normal cells with each exhibiting unique drug sensitivity spectra. Glial cells are the gluelike material surrounding neurons in the brain. In vitro assays provide an initial platform for cancer drug discovery approaches. There was also an urgent need for single cell suspensions of epidermal cells suitable for clonogenic assays 3, fluorescence activated cell sorting, and flow cytometry 3. To determine whether in vitro chemosensitivities of clones from metastases of human tumors varied, biopsy specimens of two separate metastatic lesions were obtained from 75 patients. Hematotoxicity testing by cell clonogenic assay in drug. Application of in vivo and in vitro pharmacokinetics for. Automated versus manual counting of clonogenic assays. The human tumor clonogenic assay as a model system in cell.